Gurjar Karun, Rampuria Sakshi, Joshi Uma, Palit Paramita, Bhatt K V, Mohapatra T, Sharma Ramavtar
Plant Biotechnology Centre, Swami Keshwan and Rajasthan Agricultural University, Bikaner, Rajasthan, 334006, India,
Appl Biochem Biotechnol. 2014 Aug;173(8):2116-28. doi: 10.1007/s12010-014-1011-2. Epub 2014 Jun 18.
Moth bean (Vigna aconitifolia (Jacq.) Marechal), an important grain-legume crop grown in hot desert regions of Thar, under scorching sun rays, was investigated for heat tolerance at molecular level. In the present study, we constructed a forward suppression subtractive hybridization (SSH) cDNA library of heat tolerant genotype RMO-40 to identify genes expressing under delayed response to elevated temperature. Heat induction was carried out by exposing 14-day-old seedlings to elevated temperature of 42 °C for 30 min. A total of 125 unigenes (33 contigs and 92 singletons) were derived by cluster assembly and sequence alignment of 200 ESTs; out of 125 unigenes, 21 (16 %) were found to be novel to moth bean. Gene ontology functional classification terms were retrieved for 98 (78.4 %) unigenes of which 73 (58.4 %) ESTs were functionally annotated (GO consensus) where 19 unigenes were annotated with 11 enzyme commission (EC) codes and were mapped to 25 different KEGG pathways. We have identified a majority of heat-shock proteins (constituting 35 % of the present library) aiding heat stress tolerance to moth bean. An expression level of 22 ESTs generated from the above SSH cDNA library was studied through semiquantitative RT-PCR assay simultaneously under 5 and 30 min of heat stress at 42 °C.
绿豆（Vigna aconitifolia (Jacq.) Marechal）是一种重要的谷物豆类作物，生长在塔尔沙漠炎热地区，在烈日下接受考验，本研究在分子水平上对其耐热性进行了调查。在本研究中，我们构建了耐热基因型RMO - 40的正向抑制消减杂交（SSH）cDNA文库，以鉴定在温度升高延迟响应下表达的基因。通过将14日龄幼苗暴露于42°C的高温下30分钟进行热诱导。通过对200个EST进行聚类组装和序列比对，共获得125个单基因（33个重叠群和92个单拷贝）；在125个单基因中，发现有21个（16%）对绿豆来说是新的。对98个（78.4%）单基因检索了基因本体功能分类术语，其中73个（58.4%）EST进行了功能注释（GO共识），其中19个单基因用11个酶委员会（EC）代码注释，并映射到25条不同的KEGG途径。我们已经鉴定出大多数热休克蛋白（占本文库的35%）有助于绿豆耐受热胁迫。通过半定量RT-PCR分析，同时研究了在42°C下热胁迫5分钟和30分钟时，从上述SSH cDNA文库产生的22个EST的表达水平。
